黄瓜疫病抗性基因的精细定位与候选基因克隆

Cucumber Phytophthora blight caused by Phytophthora melonis(P.melons)occures seriously in Southern China because of high temperature and humidity, and therefore it is partucularly urgent to exploit resistence gene resources, After many years for creation of cucumber germplasm resources, we obtained a cucumber inbred line, named JSH with high resistance to Phytophthora blight. The investigation of inheritance of resistance against Phytophthora melonis in JSH showed that the Phytophthora blight resistance traits are quality traits, controlled by one dominant gene. The resistance gene was mapped on cucumber chromosome 5, using the specific locus amplified fragment sequencing (SLAF-seq) method. Based on previous study results, we plan to implement finer mapping using Indel, SSR and SNP polymorphism molecular markers between parents (JSH and B80), mined by genome resequencing, and a large BC1F2 segregation population to narrow the interval. In addition, we also plan to perform transcriptome sequencing for parents respectively to analyze differential expression of genes between JSH (resistant material) and B80 (susceptible material). Finally, in order to identify and clone candidate genes for Phytophthora blight resistance, we will analyze the genes within the mapped interval by integrating the data from genome resequencing and transcriptome sequencing. So,the smooth implementation of this study will lay a foundation for the future studies on functional verification and disease resistance mechanism of the disease resistance gene, and molecular breeding of Phytophthora blight resistance of cucumber.

疫病是黄瓜重要病害之一，在全国各地均有发生，特别是华南地区高温高湿，黄瓜疫病发生尤为严重。本课题组经多年材料创制，得到1份高抗疫病华南型黄瓜自交系JSH，对其抗性性状调查结果显示在瓜类疫霉接种条件下，该性状属于显性单基因控制的质量性状。利用SLAF-seq技术将抗性基因定位于黄瓜第5号染色体。本研究拟在前期研究基础上，利用基因组重测序技术在亲本（JSH和感病材料B80）间开发Indel、SSR、SNP等多态性标记，通过BC1F2大群体对抗性基因进一步定位，缩小距离；同时，分别对疫霉菌接种前后的亲本进行转录组测序，分析抗、感材料中基因的差异表达情况；最后，综合基因组重测序和转录组数据对定位区间基因进行分析，确定并克隆疫病抗性候选基因。本研究将探明华南型黄瓜JSH疫病抗性位点并克隆抗性候选基因，为进一步确证疫病抗性基因及研究其抗病机理和黄瓜抗疫病分子育种奠定基础。

疫病是黄瓜重要的病害之一，在全国各地均有发生，特别是华南地区高温高湿，黄瓜疫病发生尤为严重。本项目组经多年材料创制，得到1份高抗疫病的华南型黄瓜自交系JSH，对其抗性性状遗传规律研究得知其抗病性状为显性单基因控制的质量性状。利用SALF-seq技术将抗性基因定位在第5号染色体。利用基因组重测序技术在亲本间开发INDEL,SSR,SNP等多态性标记，通过BC1F2群体对抗性基因进行进一步定位。同时对疫霉菌接种前后的亲本进行转录组测序，分析抗感材料中基因的差异表达情况，最后对定位区间基因进行分析，确定候选基因。本研究获得主要结果如下（1）建立了快速鉴定瓜类疫病的离体鉴定体系。（2）将抗性基因定位到黄瓜第5号染色体，将该基因克隆测序发现在定位区间内，根据69个多态性SNP位点，设计17个CAPs酶切位点，4个dCAPS酶切位点，在F2代验证，获得连锁标记Cs3419019。（3）根据生物信息学分析，确定1个候选基因，为植物防御卵菌侵染基因。对该基因进行克隆测序，发现该基因（命名为CsPmR1）在JSH黄瓜中的CDS序列的第709个核苷酸缺失，并导致其后密码子移位，产生终止密码子，使翻译提前终止。该项目的研究不仅为黄瓜乃至瓜类疫病抗性调控机理研究奠定前期基础，也为瓜类抗疫病分子育种提供理论和技术支撑。